Seroussi et al. Genet Sel Evol (2017) 49:19 DOI 10.1186/s12711-017-0296-3 Genetics Selection Evolution

RESEARCH Open Access Unveiling genomic regions that underlie differences between Afec‑Assaf sheep and its parental Awassi breed Eyal Seroussi, Alexander Rosov, Andrey Shirak, Alon Lam and Elisha Gootwine*

Abstract Background: Sheep production in Israel has improved by crossing the fat-tailed local Awassi breed with the East Friesian and later, with the Booroola Merino breed, which led to the formation of the highly prolific Afec-Assaf strain. This strain differs from its parental Awassi breed in morphological traits such as tail and horn size, coat pigmentation and wool characteristics, as well as in production, reproductive and health traits. To identify major associated with the formation of the Afec-Assaf strain, we genotyped 41 Awassi and 141 Afec-Assaf sheep using the Illumina Ovine SNP50 BeadChip array, and analyzed the results with PLINK and EMMAX software. The detected variable genomic regions that differed between Awassi and Afec-Assaf sheep (variable genomic regions; VGR) were compared to selection signatures that were reported in 48 published genome-wide association studies in sheep. Because the Afec-Assaf strain, but not the Awassi breed, carries the Booroola mutation, association analysis of BMPR1B used as the test was performed to evaluate the ability of this study to identify a VGR that includes such a major gene. Results: Of the 20 detected VGR, 12 were novel to this study. A ~7-Mb VGR was identified onOvies aries chromo- some OAR6 where the Booroola mutation is located. Similar to other studies, the most significant VGR was detected on OAR10, in a region that contains candidate genes affecting horn type RXFP2( ), climate adaptation (ALOX5AP), fiber diameter (KATNAl1), coat pigmentation (FRY) and genes associated with fat distribution. The VGR on OAR2 included BNC2, which is also involved in controlling coat pigmentation in sheep. Six other VGR contained genes that were shown to be involved in coat pigmentation by analyzing their mammalian orthologues. Genes associated with fat distribution in humans, including GRB14 and COBLL1, were located in additional VGR. Sequencing DNA from Awassi and Afec-Assaf individuals revealed non-synonymous mutations in some of these candidate genes. Conclusions: Our results highlight VGR that differentiate the Awassi breed from the Afec-Assaf strain, some of which may include genes that confer an advantage to Afec-Assaf and Assaf over Awassi sheep with respect to intensive sheep production under Mediterranean conditions.

Background Awassi dairy strain [2]. The Assaf dairy breed was formed In Israel, within-breed selection, crossbreeding and gene about 30 years later by crossing the improved Awassi introgression have contributed to the transition of the with the European East Friesian (EF) breed [3]. In 1986, sheep industry from traditional extensive production breeding of the highly prolific Afec-Assaf strain was ini- using the native fat-tailed local Awassi breed to highly tiated by introgressing the Booroola mutation (B) at the intensive production with the Assaf and Afec-Assaf BMPR1B gene into the Assaf breed by crossing Assaf sheep [1]. Selection within the local Awassi sheep that ewes with Booroola Merino rams [4–6]. The high prolifi- began in the 1930s led to the formation of the improved cacy of the Afec-Assaf strain is due to the presence of the B allele, which is inherited in an almost completely domi- nant mode [7], since BB and B ewes have similar pro- *Correspondence: [email protected] + Institute of Animal Science, ARO, The Volcani Center, PO Box 15159, lificacy. However, since the perinatal lamb survival rate is 7528809 Rishon LeZion, Israel higher in B+ than in BB ewes, B+ is the recommended

© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Seroussi et al. Genet Sel Evol (2017) 49:19 Page 2 of 10

genotype for females in commercial flocks [7]. TheB By using the ovine single nucleotide polymorphism allele segregates in the Afec-Assaf population, thus gen- (SNP) 50 BeadChip array (Illumina Inc., San Diego, CA) otyping lambs for the Booroola mutation and selecting and BMPR1B as a test gene, the aims of our study were only the B+ ewe lambs as replacements is a common to: (1) compare the Awassi and Afec-Assaf genomes, practice in Afec-Assaf flocks to maintain high prolificacy searching for variable genomic regions (VGR) that differ in the long term. Afec-Assaf sheep differ widely from the between the two breeds; and (2) link these VGR to genes Awassi sheep in a number of phenotypic characteristics, and selection signatures that were previously described such as the presence of horns, tail shape, coat color pig- in sheep GWAS. mentation and wool quality, as well as seasonality, prolifi- cacy, and milk production [8, 9] (Table 1). Methods In sheep, genome-wide association studies (GWAS) Ethics statement have contributed to better understanding of their domes- Experimental protocols were approved (Approval No. IL tication [10, 11], as well as to the identification of specific 415-12) by the Volcani Center Institutional Animal Care genomic regions that are associated with differences in and Use Committee. various traits between breeds (see Additional file 1: Table S1). GWAS have made it possible to identify candidate Animals genes that underlie various phenotypic differences, for Local Awassi sheep are raised by Bedouin growers in instance in Texel sheep RXFP2 for horn type [12] and small unconnected flocks that are maintained under disease-resistance genes such as PITX3 and DMP1 which extensive traditional management with no records. The are involved in microphthalmia [13] and in Corriedale Awassi cohort consisted of 24 local Awassi rams that sheep for rickets [14]. included all of the rams from five flocks, and 17 improved Cryptic relatedness, which occurs when there is Awassi ewes from the Ein-Harod flock. Since the degree unknown kinship within the sample, and population of relationship between animals from the same flock stratification due to random genetic drift in the sam- is unknown, the effective number of local Awassi indi- ple’s subpopulations are two major confounding effects viduals might be smaller than sampled. However, this that cause spurious associations in GWAS analyses [15]. was not the case, since a genomic relationship analysis Hence, incorporating a known genetic marker that is (see Additional file 2: Table S2) showed no strong kin- located within a major gene that segregates within the ship between individuals within or among flocks. The tested population into the GWAS analysis may assist Afec-Assaf cohort consisted of 22 males and 119 females in verifying the power of the study for detecting selec- from the experimental flock of the Volcani Center at Bet tion signatures. This has been the case in several sheep Dagan and two commercial farms. Genotyping indicated GWAS, in which the MC1R gene that affects coat pig- that 35, 95 and 11 animals of the Afec-Assaf cohort were mentation [16] was used as the ‘test’ gene, and poly- homozygous BB, heterozygous B+ or non-carrier ++ for morphism at the GDF8 gene associated with muscle the Booroola mutation, respectively. As expected [7], the hypertrophy [17] was used to establish criteria to detect average prolificacy of the BB and B+ females (based on 3 selection signatures. to 9 parity records per animal) was high, i.e. 3.1 and 3.0

Table 1 Phenotypic, production and reproductive traits of local Awassi, improved Awassi, Assaf and Afec-Assaf sheep Traits Local Awassi Improved Awassi Assaf Afec-Assaf

Body size Smalla Largea Largea Largea Tail shape Fat tail Fat tail Partial fat tail Partial fat tail Horn shape Most rams and ewes are Most males and females are polled, horned or carry scurs but some carry short knobs or scurs Wool type Coarse Coarse Medium-coarse Medium-coarse Coat pigmentation Most sheep have brown Most sheep have white face face and white fleece and white fleece Seasonality Seasonal Seasonal Moderately seasonal Moderately seasonal Adaptation to Middle-Eastern High High Intermediate Intermediate climate Milk production Low High High High Prolificacy (lambs born/ewe 1.2 1.3 1.6 2.5 lambing) a Small: ram body weight ~70 kg; large: ram body weight ~110 kg Seroussi et al. Genet Sel Evol (2017) 49:19 Page 3 of 10

lambs born/lambing, respectively. No lambing records Identification of candidate polymorphisms were available for non-carrier ++ females. Exon sequences of the candidate genes were downloaded from NCBI (https://www.ncbi.nlm.nih.gov) and genomic Genotyping using the ovine 50 K beadchip reads were mapped to these templates using GAP5 soft- DNA was extracted from blood samples using the ware [20]. BWA options for this mapping were set to bam DNeasy Blood & Tissue Kit (Qiagen, Valencia, CA) bwasw -t 8 -T 30 [21]. Genetic markers were identified according to the manufacturer’s instructions. DNA sam- and called by comparing the consensus contig sequences ples were genotyped by the Ovine SNP50 BeadChip of the Awassi and Assaf individuals with the GAP5 con- assay using standard procedures (http://www.illumina. tig comparator and the heterozygosity search option com). All the Awassi and some of the Afec-Assaf sam- (value = 10). ples were genotyped as part of the International Sheep Genomic Consortium initiative (http://www.sheephap- Scoring amino acid substitutions map.org). Other Afec-Assaf samples were genotyped Amino acid substitutions were evaluated using at the Centre for Reproduction and Genomics (CRG), PROVEAN analysis v1.1 [22] which scores the effect of Invermay, New Zealand. a variation in sequence on the function of that Of the 59,454 SNPs on the ovine SNP50 BeadChip, protein. 46,563 SNPs that were randomly distributed across the genome passed the quality-control filter criteria (call rate Statistical analysis higher than 99%, genotyping frequency higher than 95%, Various methods have been applied to verify selection minor allele frequency lower than 0.05 and Hardy–Wein- signatures in cattle [23] as well as in sheep. To minimize berg equilibrium P > 0.001), and were used for further the risk of detecting spurious associations in our GWAS analyses. analysis, we applied two tools: PLINK, which is suitable for calculating either Fst values or probability values for Genotyping at the RXFP2 gene genetic association test and EMMAX that calculates Phenotypic variation in horn appearance includes the these probability values using a relationship matrix that presence of normal horns (in females they are smaller accounts for relatedness within the sample. Association than in males), deformed horns (scurs), short knobs between SNPs and breed (Awassi or Afec-Assaf) were at the site of horn growth, and a polled-non-horned evaluated using PLINK v1.90b3.38 64-bit [24]. Probabil- growth phenotype, which may include a concave ity (Fisher’s exact test of significance) and Fst values for depression in the skull bone at the horn site [18]. DNA differences in allele frequency were estimated with the from Awassi and Afec-Assaf sheep with different horn following options on the command line: –sheep –hwe phenotypes was extracted from blood samples or buc- 0.001 –geno 0.1 –maf 0.05 –fst –assoc fisher. Fst estimates cal swabs using standard DNA-extraction protocols. were obtained only for the autosomes and were com- Primers, PCR conditions and separation of PCR prod- puted using the method introduced by Weir and Cocker- ucts on an agarose gel to genotype animals that carry ham. The Efficient Mixed-Model Association eXpedited a 1833-bp genomic insertion located in the 3′-UTR of (EMMAX) program [25] was used to identify genomic RXFP2, were performed according to Wiedemar and regions on autosomes and on the X . For the Drogemuller [19]. Association between the genotype at EMMAX analysis, the following options were used on the RXFP2 gene and horn appearance was tested by Chi the command lines: emmax-kin -v -h -s -d 10; emmax -v square analysis. -d 10, for the creation of the identity by state (IBS) rela- tionship matrix and for the association test, respectively. DNA sequencing Genomic inflation factors were equal to 9.35 and 1.34 To obtain preliminary information on variation of the for the unadjusted PLINK Fisher and EMMAX analy- coding regions of genes that are included in VGR, DNA ses, respectively. Using each of the programs, SNPs were of one Afec-Assaf individual and one Awassi individual, ranked according to their association with one of the selected randomly, was sequenced using the Illumina breeds by chance. The top ~0.1% (n = 50) significant 100-bp paired-end technology (HiSeq 2000, Illumina) SNPs and their adjacent neighboring SNPs, which ranked with one or two lanes, respectively. The sequences were in the top 0.5% and were found within 1.5 Mb upstream deposited in the European Nucleotide Archive (ENA) or downstream of the top marker, were used to define under accession number PRJEB12018. Achieved coverage VGR. Neighboring genomic regions were combined into was 11- and 26-fold for the Afec-Assaf and Awassi indi- one region if they overlapped. The Bonferroni-corrected −6 viduals, respectively. significance threshold of 1.07 × 10 (0.05/46,563) was Seroussi et al. Genet Sel Evol (2017) 49:19 Page 4 of 10

used to correct for multiple comparisons and the thresh- Variable genomic regions on OAR6 (between 24.97 old for significance was set at P < 0.05. The position of and 32.04 Mb) regions was determined according to version 3.1 of the As expected, a VGR (#10) that spanned the BMPR1B sheep genome assembly. gene (between positions 29.36 and 29.45 Mb) was iden- tified on OAR6 (Table 2), with the most significant SNP, Bioinformatics s24937.1, being located about 3.0 Mb upstream of the Candidate regions were examined using the sheep gene. None of the four SNPs of the Illumina Ovine SNP50 genome browsers Build v3.1 [26–28]. We used the fol- BeadChip that are located within non-coding regions of lowing databases: NCBI [29], GeneCards® [30], and BMPR1B namely: OAR6_33210385.1, OAR6_33229928.1, SheepQTL [31], as well as literature searches, to obtain OAR6_33291928.1 or s21306, reached significance gene annotations and functions. (P > 0.05). DNA sequencing showed that the Afec-Assaf individual was heterozygous for the Booroola mutation, Results and discussion while the Awassi individual did not carry the mutation. PLINK analysis showed a correlation of 0.98 between the A similar level of precision in the detection of a point values of Fst and the minus logarithm of the probability mutation was obtained in a GWAS applied in Texel sheep according to Fisher’s exact test (data not shown). Thus, to map the causative mutation for microphthalmia, which since both parameters yielded similar estimates for allele revealed a region of about 2.6 Mb [13]. In the case of the frequency differences, we chose to consider only theF st autosomal recessive lethal disorder brachygnathia, cardio- values in the further analyses. PLINK and EMMAX- megaly and renal hypoplasia syndrome in polled Merino based GWAS revealed 36 and 32 VGR, respectively (see sheep, SNPs that are associated with this syndrome are Additional file 3: Table S3). Fst values for the most signifi- spread across 1.1 Mb [32]. However, the precision that was cant SNPs in each region in the PLINK analysis ranged achieved in a GWAS for detecting the causative mutation from 0.46 to 0.80, and the corresponding P values in the for dominant pigmentation in the MC1R gene was higher EMMAX analysis ranged from <10−5 to <10−15. The 20 [16], with only one significant SNP that was 25 kb away VGR that were detected by both PLINK and EMMAX from MC1R, being identified for the Manchega sheep pop- analyses were further analyzed (Table 2 and Fig. 1). ulation, in which this trait segregates.

Table 2 Variable genomic regions detected in Awassi and Afec-Assaf sheep according to both PLINK and EMMAX analy- ses

Variable genomic OAR number Region position PLINK - Peak SNP EMMAX -Peak SNP PLINK Fst EMMAX region number (Mb) position (Mb) position (Mb) P value

1 1 2.72 2,721,556 0.61 1.0e 07 − 2 99.83–101.28 99,832,319 0.65 5.4e 08 − 3 103.40–103.45 103,452,513 0.60 2.5e 08 − 4 164.60 164,604,667 0.59 6.7e 07 − 5 266.24 266,241,278 0.55 5.3e 07 − 6 2 83.11–84.53 83,214,641 84,054,194 0.59 1.0e 05 − 7 145.65–148.41 148,406,594 145,648,041 0.68 3.9e 07 − 8 3 134.23–134.49 134,234,489 0.66 6.7e 07 − 9 6 20.99–24.00 22,323,737 0.66 5.1e 08 − 10 24.97–32.04 26,058,630 0.73 1.8e 10 − 11 54.26–55.67 54,261,462 0.55 5.8e 06 − 12 7 96.99 96,995,379 0.58 2.9e 07 − 13 8 62.62–62.83 62,735,336 0.57 6.8e 08 − 14 78.36–80.60 78,364,891 80,605,889 0.67 5.2e 09 − 15 10 27.65–31.64 30,700,853 0.80 8.4e 15 − 16 33.75–36.24 36,238,012 33,752,924 0.66 1.2e 07 − 17 17 8.13–8.74 8,739,357 0.66 1.9e 06 − 18 20 37.51 37,515,740 0.57 2.5e 06 − 19 25 6.65–8.66 7,392,689 0.59 4.7e 08 − 20 26 36.58 36,580,518 0.58 5.4e 06 − Seroussi et al. Genet Sel Evol (2017) 49:19 Page 5 of 10

a

b

Fig. 1 Manhattan plot of GWAS comparing Awassi and Afec-Assaf sheep

The VGR on OAR6 that includes the BMPR1B gene Awassi and Afec-Assaf sheep (see Additional file 4: Fig- also emerged in a GWAS that searched for genomic dif- ure S1), to identify those that carried a 1833-bp genomic ferences between dairy and non-dairy sheep breeds [17], insertion located in the 3′-UTR of RXFP2. It has been and in another study that focused on quantitative trait shown that this insertion is linked to horn inheritance in loci (QTL) for carcass traits [33]. Since the Booroola Swiss sheep breeds [19]. Our results (see Additional file 5: mutation was introgressed into the Assaf breed from Table S4) show that in Awassi and Afec-Assaf sheep, there the small-size non-dairy Booroola Merino breed, the is also an association between the RXFP2 polymorphism genomic difference that was found between the Awassi in the 3′-UTR and the hornness-polledness phenotype and Assaf breeds at around 29 Mb on OAR6 might also (P < 0.0001), with absence of the insertion being associated be related to the difference in allelic frequency of genes with the horned phenotype. However, the range of pheno- that control these traits. One relevant gene was ABCG2, types observed in the group of heterozygous animals indi- which has a major effect on milk production, and is cated that other genetic modifications may also underlie mapped to OAR6 at 36.5 Mb, about 3.0 Mb downstream the horned phenotype. A recent study of 38 sheep breeds to the 3′ end of that VGR [17]. [40] reached similar conclusions i.e. that the 1.78-kb inser- tion in the 3′-UTR of RXFP2 cannot be considered as the Variable genomic regions on OAR10 that span 30 Mb only cause of polledness in sheep. A major VGR (#15) was found on OAR10 (between posi- tions 27.65 and 31.64 Mb, Table 2). Significant selec- Climate adaptation tion signatures in this chromosomal region have been The EF and Awassi breeds—the parental breeds of the detected in several GWAS in which sheep breeds were Afec-Assaf—differ in their adaptability to the hot and compared for horn phenotype, climate adaptation, fiber humid Mediterranean climate. Susceptibility to pneu- diameter, coat pigmentation or fat distribution [10–12, monia in the summer is a major death threat in Israel 18, 34–39]. for pure EF but not Awassi lambs [41]. A GWAS that investigated environmental adaptive selection in sheep Horn phenotype [36] suggested ALOX5AP (on OAR10 between 30.36 and Awassi rams are mostly horned, as well as some of the 30.38 Mb) as a candidate gene for climate adaptation. ewes, whereas most Assaf sheep are polled, similar to the Located in the distal part of VGR #15 on OAR10, this situation observed in the EF parental breed. Indeed, the gene encodes a protein that is required for the synthesis RXFP2 gene (on OAR10 between 29.45 and 29.50 Mb), of leukotrienes, which are biologically active lipid media- which is involved in horn inheritance [12], was identified tors involved in various types of inflammatory responses. in the middle of this OAR10 VGR (Table 2). To further cor- In humans, polymorphism in ALOX5AP has been associ- roborate this, we genotyped 61 horned and non-horned ated with lung function [42, 43]. Seroussi et al. Genet Sel Evol (2017) 49:19 Page 6 of 10

Fiber diameter fat distribution: carrying a fat tail was associated with a In a GWAS that included Chinese sheep breeds, differ- decrease in the relative amount of body fat (omental, ences in the VGR on OAR10 were associated with differ- mesenteric, kidney, channel and scrotal fat), as well as ences in the coefficient of variation of fiber diameter [37]. a decrease in the relative amount of subcutaneous and Indeed, the Awassi and Assaf breeds differ in their varia- intramuscular fat. tion in fiber diameter, with the Awassi’s fibers being more GWAS comparing fat- and thin-tailed sheep [38, 39, uniform [44]. TheKATNAL1 gene (on OAR10 between 47, 48] or studies on differential in such 30.77 and 30.78 Mb) was the closest gene mapped to the breeds [49–51] revealed several chromosomal regions most significant SNP (OAR10: 30,700,856). that are associated with the presence of a fat tail (see Additional file 9: Table S6). Only two of these regions, Coat pigmentation i.e. regions numbered 14 and 15, both on OAR10, over- As indicated in Table 1, the coat color of most Afec- lapped with VGR that distinguish Awassi from Afec- Assaf sheep is white, while most Awassi sheep have Assaf sheep. These two regions were identified in a study reddish-brown pigmentation on their head, neck and that compared Cyprus fat-tailed sheep, a breed related to legs. The mode of inheritance of coat pigmentation pat- the Awassi breed, and Laticauda sheep, to several Italian tern in sheep is complex, as already shown for the pie- thin-tailed breeds [39]. bald phenotype in Merino sheep [45]. Of the 11 genes Taken together, the VGR on OAR10 that spans a region that have been suggested as candidates that affect sheep between 27.65 and 31.64 Mb and detected in the current coat pigmentation (see Additional file 6: Table S5), FRY study, has been associated in various sheep GWAS with (on OAR10 between 28.98 and 29.19 Mb) was located several phenotypic traits that differ between Awassi and in the middle of VGR #15 on OAR10. FRY is a key can- Afec-Assaf sheep. didate gene for the piebald phenotype in Merino sheep [45] and for coat color differences between Rambouillet Additional variable genomic regions associated (white coat color) and Suffolk (black head and legs) sheep with differences in coat pigmentation [35], and is associated with the black spot phenotype in Two of the coat pigmentation-related genes known in Valley-type Tibetan sheep [38]. Thus, polymorphism sheep: BNC2 and TYRP1 (see Additional file 6: Table S5) associated with the FRY gene may contribute to the dif- were localized, respectively, within or close to VGR #6 ferences in coat color pigmentation distribution between that spans a region between 83.11 and 84.53 Mb on OAR2 the Awassi and Afec-Assaf sheep. (Table 2). BNC2 (on OAR2 between 84.29 and 84.69 Mb) was identified as a candidate gene for coat pigmentation Sequence analysis in Italian breeds [11] with the most differentiated breed Comparing the coding sequences of the three candi- being the Comisana, which resembles the Awassi breed date genes, namely: ALOX5AP, KATNAL1 and FRY, of with a white body and brick-red face. The other breeds Awassi and Assaf individuals revealed two synonymous that contrasted with the Comisana were the Altamurana mutations, no sequence variation and three conserva- and Leccese white-coat breeds, and the Sardinian ances- tive amino-acid substitutions: p.V383I, p.V1207I and tral black sheep. Notably, the TYRP1 gene (on OAR2 p.A1722S, respectively (see Additional file 7). between 80.60 and 80.62 Mb), which is associated with skin and fiber pigmentation differences between Awassi Fat distribution and Merino sheep [52], and with pigmentation poly- While the Awassi sheep have a relatively large fat tail, morphism in Soay sheep [53] and in Finnsheep [54], was which usually hangs down to the hock, most Assaf and mapped closely to VGR #6 (on OAR2 between 83.11 and Afec-Assaf sheep carry a reduced-size fat tail which var- 84.53). Comparing sequences of Awassi and Afec-Assaf ies in size (see Additional file 8: Figure S2). Archeologi- BNC2- and TYRP1-coding regions (see Additional file 7) cal evidence has documented the presence of fat-tailed yielded one neutral substitution (p.W70R) and one con- sheep in the Fertile Crescent as early as 4000 years BC. servative replacement, respectively. In this region, sheep were the major source of animal fat, To further search for possible candidate genes associ- which was consumed as a delicacy and used for indus- ated with coat pigmentation differences between Awassi trial purposes. Selection for fat tail was driven, among and Afec-Assaf sheep, we compiled a list of 172 genes other reasons, by the convenience of harvesting the fat known to affect coat pigmentation in humans [55–57], tissue from the carcasses. A study of body and carcass cattle [58] and other mammals [59] (see Additional conformation of Awassi and Merino–Awassi crossbred file 10: Table S7). Nine of these genes namely: NTRK1, sheep [46] demonstrated that the reduction in fat-tail RPL24, SEMA4A, KRT1, KRT4, KRT75, BMPR1B, size in Awassi crosses is associated with a change in body MAB21L2, and EDARADD were located within, or Seroussi et al. Genet Sel Evol (2017) 49:19 Page 7 of 10

close to one of the VGR that were identified on six ovine Twelve of the 20 VGR identified in the current study, . namely VGR #1, 2, 3, 4, 5, 7, 8, 9, 11, 12, 17 and 18 Taken together, our results indicate that two of the coat (Table 2) were not described in any of the 48 GWAS pigmentation-related genes known in sheep: FRY and sheep studies listed in Table S1 (see Additional file 1: BNC2, and probably TYRP1, as well as some other genes Table S1). These VGR, which may have arisen due to have possible roles in the differences in coat pigmenta- either selection or random genomic drift, may contain tion between Awassi and Afec-Assaf sheep. chromosomal regions that contribute to the genetic dif- ferences between the Awassi breed and its derived Afec- Additional variable genomic regions associated Assaf strain. with differences in fat distribution Fat distribution between subcutaneous and visceral adi- Conclusions pose tissue in humans is genetically controlled [60]. After Using the Illumina Ovine SNP50 BeadChip array, we annotating 107 genes associated with fat distribution in revealed 20 VGR that are associated with differences humans [60–64] (see Additional file 11: Table S8), we in morphological, production and reproductive traits found that the DCST2 gene was annotated close to VGR between the Afec-Assaf strain and the Awassi breed, #3 on OAR1 (between 103.40 and 103.45 Mb), and that a hardy Middle Eastern fat-tailed breed. Focusing on two genes, GRB14 and COBLL1, were both located close BMPR1B as a test gene allowed us to evaluate the abil- to VGR #7 on OAR2 (between 145.65 and 148.41 Mb). ity of our study to identify VGR that harbor major Only a few synonymous and conservative substitu- genes. We compared our results to those of 48 GWAS tions were identified in DCST2 and GRB14, respectively, in sheep, and GWAS in other domestic animals and when the corresponding coding sequences of Awassi in humans. Thus, we were able to highlight candidate and Afec-Assaf sheep were compared (see Additional genes that are involved in differences between Awassi file 7). However, in the case of COBLL1, four non-synon- and Afec-Assaf sheep regarding morphological traits, ymous substitutions were found, one of which, p.I448S such as horn appearance, presence of a fat tail and coat (ref XP_012011424.1), was deleterious according to the pigmentation. In most cases, a comparison between the PROVEN analysis [22]. If COBLL1 is indeed involved in exon sequences of the candidate genes from Awassi and the control of differences in fat deposition in sheep, fat- Afec-Assaf individuals revealed synonymous or neutral tailed sheep could serve as a natural large animal model variations. for studying the possible adverse metabolic and physi- It is of interest that 60% of the VGR identified in the ological outcomes of body fat distribution. current study were not described in previous sheep GWAS that included a variety of sheep breeds. Bearing Variable genomic regions not associated with a specific in mind that VGR may also arise from random drift, trait selection for genes that are included in these VGR may Our analysis revealed additional VGR that were previ- have further improved the Assaf breed and contrib- ously identified in other GWAS. However, these were uted to establish it as the major dairy breed in Israel not associated with a specific production, reproductive and Spain, where it outperforms the local dairy breeds or health trait. VGR #13 on OAR8 (between 62.62 and [66]. 62.83 Mb) was detected in a GWAS that included mul- tiple worldwide breeds [10], while VGR #16 on OAR10 (between 33.75 and 36.24 Mb) was detected in a GWAS Additional files that comprised five American breeds [35]. VGR #19 on OAR25 (between 6.65 and 8.66 Mb) was characterized Additional file 1: Table S1. Genome-wide association studies in sheep reviewed in the current study [10–14, 17, 18, 32–39, 47, 48, 54, 65, 67–95]. as an “ancestral signature of selection” that differentiates Additional file 2: Table S2. Genomic relationship within and between European from Mediterranean breeds early in domes- local Awassi flocks. tication [11]. It is worth noting that the EF and Awassi Additional file 3: Table S3. Variable genomic regions (VGR) that differ breeds, which are the parental breeds of the Afec-Assaf between the Awassi and the Afec-Assaf according to PLINK and EMMAX strain, belong to these two groups, respectively. This VGR analyses. SNPs were ranked according to their P-values, and the selected regions included the top 0.1% markers and their neighboring (1.5 Mb on OAR25 was also identified in several studies in which upstream and 1.5 Mb downstream) markers ranked in the top 1% of the different breeds were compared [10, 23, 38]. Finally, VGR markers. Two or more genomic regions were combined if they over- #20 on OAR26 (36.58 Mb) was identified in a GWAS that lapped. Region position was determined using sheep genome assembly analyzed the highly prolific Afec-Assaf ewes with differ- version 3.1. Regions identified by both analyses are marked in gray. ent perinatal lamb viability rates [65]. Seroussi et al. Genet Sel Evol (2017) 49:19 Page 8 of 10

that is expressed in both oocytes and granulosa cells. Biol Reprod. Additional file 4: Figure S1. Different horn phenotypes in Awassi (A 2001;64:1225–35. to D) and Afec-Assaf (E to H) sheep. (A) Polled, female. (B) Knobs, female. 7. Gootwine E, Reicher S, Rosov A. Prolificacy and lamb survival at birth in (C) Horns, female. (D) Horns, male. (E) Polled, female. (F) Knobs, male. (G) Awassi and Assaf sheep carrying the FecB (Booroola) mutation. Anim Knobs, male. (H) Scurs, male. Reprod Sci. 2008;108:402–11. Additional file 5: Table S4. Association between presence of a 1.8-kb 8. Gootwine E, Pollott GE. Factors affecting milk production in Improved insertion at the 3′-UTR of RXFP2 and horn phenotype in Awassi and Afec- Awassi dairy ewes. Anim Sci. 2000;71:607–15. Assaf sheep. A 428-bp PCR product indicates the presence of the insertion 9. Pollott GE, Gootwine E. Reproductive performance and milk produc- [19]. tion of Assaf sheep in an intensive management system. J Dairy Sci. 2004;87:3690–703. Additional file 6: Table S5. Candidate genes for sheep coat colors and 10. Kijas JW, Lenstra JA, Hayes B, Boitard S, Porto Neto LR, San Cristobal patterns [10, 11, 16, 34, 35, 38, 45, 52–54, 96, 97]. M, et al. Genome-wide analysis of the world’s sheep breeds reveals Additional file 7. Candidate gene sequences. This file contains high levels of historic mixture and strong recent selection. PLoS Biol. sequences of candidate genes in a FASTA-like format. Each sequence 2012;10:e1001258. header consists of the gene symbol followed by its reference GenBank 11. Fariello MI, Servin B, Tosser-Klopp G, Rupp R, Moreno C, San Cristobal M, accession number and the breed name. Polymorphic nucleotide positions et al. Selection signatures in worldwide sheep populations. PLoS One. are indicated in blue and red font colors. 2014;9:e103813. 12. Johnston SE, McEwan JC, Pickering NK, Kijas JW, Beraldi D, Pilkington JG, Additional file 8: Figure S2. Variability in fat-tail phenotypes in Assaf et al. Genome-wide association mapping identifies the genetic basis of sheep. Awassi-like fat tail (A). Fat tails with different amounts of fat deposi- discrete and quantitative variation in sexual weaponry in a wild sheep tion and different lengths (B to H). population. Mol Ecol. 2011;20:2555–66. Additional file 9: Table S6. Candidate genes and SNP markers associ- 13. Becker D, Tetens J, Brunner A, Burstel D, Ganter M, Kijas J, et al. Micro- ated with carrying fat tail in sheep [38, 39, 47, 49–51]. phthalmia in Texel sheep is associated with a missense mutation in the Additional file 10: Table S7. Genes known to affect coat pigmentation paired-like homeodomain 3 (PITX3) gene. PLoS One. 2010;5:e8689. in humans, cattle and other mammals [55–58]. 14. Zhao X, Dittmer KE, Blair HT, Thompson KG, Rothschild MF, Garrick DJ. A novel nonsense mutation in the DMP1 gene identified by a genome- Additional file 11: Table S8. Human genes associated with fat distribu- wide association study is responsible for inherited rickets in Corriedale tion [61, 63, 64, 98–100]. sheep. PLoS One. 2011;6:e21739. 15. Astle W, Balding DJ. Population structure and criptic relatedness in genetic association studies. Stat Sci. 2009;24:451–71. 16. Kijas JW, Serrano M, McCulloch R, Li Y, Salces Ortiz J, Calvo JH, et al. Authors’ contributions Genomewide association for a dominant pigmentation gene in sheep. EG and ES designed the project. AR was responsible for breeding and data J Anim Breed Genet. 2013;130:468–75. collection for Afec-Assaf sheep. AL, AR and EG were responsible for collecting 17. Gutierrez-Gil B, Arranz JJ, Pong-Wong R, Garcia-Gamez E, Kijas J, Wiener the DNA samples. ES was responsible for all bioinformatic analyses. AS carried P. Application of selection mapping to identify genomic regions associ- out the genotyping. EG drafted the manuscript, and ES and AS contributed ated with dairy production in sheep. PLoS One. 2014;9:e94623. to the manuscript in its final version. All authors read and approved the final 18. Dominik S, Henshall JM, Hayes BJ. A single nucleotide polymorphism manuscript. on chromosome 10 is highly predictive for the polled phenotype in Australian Merino sheep. Anim Genet. 2012;43:468–70. Acknowledgements 19. Wiedemar N, Drogemuller C. A 1.8-kb insertion in the 3′-UTR Contribution from the Agricultural Research Organization, Institute of Animal of RXFP2 is associated with polledness in sheep. Anim Genet. Science, Bet-Dagan, Israel, is acknowledged. 2015;46:457–61. 20. Bonfield JK, Whitwham A. Gap5–editing the billion fragment sequence Competing interests assembly. Bioinformatics. 2010;26:1699–703. The authors declare that they have no competing interests. 21. Li H, Durbin R. Fast and accurate long-read alignment with Burrows– Wheeler transform. Bioinformatics. 2010;26:589–95. Received: 20 June 2016 Accepted: 6 February 2017 22. Choi Y, Sims GE, Murphy S, Miller JR, Chan AP. Predicting the functional effect of amino acid substitutions and indels. PLoS One. 2012;7:e46688. 23. Randhawa IA, Khatkar MS, Thomson PC, Raadsma HW. A meta-assembly of selection signatures in cattle. PLoS One. 2016;11:e0153013. 24. Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MA, Bender D, et al. References PLINK: a tool set for whole-genome association and population-based 1. Gootwine E. Mini review: breeding Awassi and Assaf sheep for diverse linkage analyses. Am J Hum Genet. 2007;81:559–75. management conditions. Trop Anim Health Prod. 2011;43:1289–96. 25. Kang M, Sul JH, Service SK, Zaitlen NA, Kong SY, Freimer NB, et al. Vari- 2. Epstein H. The Awassi sheep with special reference to the improved ance component model to account for sample structure in genome- dairy type. FAO Animal production and health paper 57. Rome: Food wide association studies. Nat Genet. 2010;42:348–54. and Agriculture Organization of the United Nations; 1985. 26. http://www.livestockgenomics.csiro.au/sheep/oar3.1.php. 3. Gootwine E, Goot H. Lamb and milk production of Awassi and East- 27. http://genome.ucsc.edu. Accessed 14 June 2016. Friesian sheep and their crosses under Mediterranean environment. 28. http://asia.ensembl.org. Accessed 14 June 2016. Small Rumin Res. 1966;20:255–60. 29. http://www.ncbi.nlm.nih.gov/. Accessed 14 June 2016. 4. Mulsant P, Lecerf F, Fabre S, Schibler L, Monget P, Lanneluc I, et al. 30. http://www.genecards.org/. 14 June 2016. Mutation in bone morphogenetic protein receptor-IB is associated with 31. http://sphinx.vet.unimelb.edu.au/QTLdb/. 14 June 2016. increased ovulation rate in Booroola Merino ewes. Proc Natl Acad Sci 32. Shariflou MR, Wade CM, Kijas J, McCulloch R, Windsor PA, Tammen I, USA. 2001;98:5104–9. et al. Brachygnathia, cardiomegaly and renal hypoplasia syndrome 5. Souza CJ, MacDougall C, Campbell BK, McNeilly AS, Baird DT. The Boo- (BCRHS) in Merino sheep maps to a 1.1-megabase region on ovine roola (FecB) phenotype is associated with a mutation in the bone mor- chromosome OAR2. Anim Genet. 2013;44:231–3. phogenetic receptor type 1 B (BMPR1B) gene. J Endocrinol. 2001;169:R1–6. 33. Matika O, Riggio V, Anselme-Moizan M, Law AS, Pong-Wong R, 6. Wilson T, Wu XY, Juengel JL, Ross IK, Lumsden JM, Lord EA, et al. Archibald AL, et al. Genome-wide association reveals QTL for growth, Highly prolific Booroola sheep have a mutation in the intracellular bone and in vivo carcass traits as assessed by computed tomography in kinase domain of bone morphogenetic protein IB receptor (ALK-6) Scottish Blackface lambs. Genet Sel Evol. 2016;48:11. Seroussi et al. Genet Sel Evol (2017) 49:19 Page 9 of 10

34. Randhawa IA, Khatkar MS, Thomson PC, Raadsma HW. Composite 55. Han J, Kraft P, Nan H, Guo Q, Chen C, Qureshi A, et al. A genome-wide selection signals can localize the trait specific genomic regions in multi- association study identifies novel alleles associated with hair color and breed populations of cattle and sheep. BMC Genet. 2014;15:34. skin pigmentation. PLoS Genet. 2008;4:e1000074. 35. Zhang L, Mousel MR, Wu X, Michal JJ, Zhou X, Ding B, et al. Genome- 56. Sulem P, Gudbjartsson DF, Stacey SN, Helgason A, Rafnar T, Magnusson wide genetic diversity and differentially selected regions among KP, et al. Genetic determinants of hair, eye and skin pigmentation in Suffolk, Rambouillet, Columbia, Polypay, and Targhee sheep. PLoS One. Europeans. Nat Genet. 2007;39:1443–52. 2013;8:e65942. 57. Sturm RA. Molecular genetics of human pigmentation diversity. Hum 36. Lv FH, Agha S, Kantanen J, Colli L, Stucki S, Kijas JW, et al. Adapta- Mol Genet. 2009;18:R9–17. tions to climate-mediated selective pressures in sheep. Mol Biol Evol. 58. Gutierrez-Gil B, Arranz JJ, Wiener P. An interpretive review of selective 2014;31:3324–43. sweep studies in Bos taurus cattle populations: identification of unique 37. Wang Z, Zhang H, Yang H, Wang S, Rong E, Pei W, et al. Genome-wide and shared selection signals across breeds. Front Genet. 2015;6:167. association study for wool production traits in a Chinese Merino sheep 59. Color genes. 2014. http://www.espcr.org/micemut. Accessed 14 June population. PLoS One. 2014;9:e107101. 2016. 38. Wei C, Wang H, Liu G, Wu M, Cao J, Liu Z, et al. Genome-wide analysis 60. Schleinitz D, Böttcher Y, Blüher M, Kovacs P. The genetics of fat distribu- reveals population structure and selection in Chinese indigenous tion. Diabetologia. 2014;57:1276–86. sheep breeds. BMC Genomics. 2015;16:194. 61. Wagner R, Machicao F, Fritsche A, Stefan N, Haring HU, Staiger H. The 39. Moioli B, Pilla F, Ciani E. Signatures of selection identify loci associated genetic influence on body fat distribution. Drug Discov Today Dis with fat tail in sheep. J Anim Sci. 2015;93:4660–9. Mech. 2013;10:e5–13. 40. Lühken G, Krebs S, Rothammer S, Küpper J, Mioč B, Russ I, et al. The 62. Shungin D, Winkler TW, Croteau-Chonka DC, Ferreira T, Locke AE, Mägi 1.78–kb insertion in the 3′–untranslated region of RXFP2 does not R, et al. New genetic loci link adipose and insulin biology to body fat segregate with horn status in sheep breeds with variable horn status. distribution. Nature. 2015;518:187–96. Genet Sel Evol. 2016;48:78. 63. Heid IM, Jackson AU, Randall JC, Winkler TW, Qi L, Steinthorsdottir V, 41. Shimshony A. Pneumonia in East-Friesian lambs and crosses in Israel. et al. Meta-analysis identifies 13 new loci associated with waist-hip ratio Rev Sci Tech Off Int Epiz. 1983;2:467–71. and reveals sexual dimorphism in the genetic basis of fat distribution. 42. Duroudier NP, Tulah AS, Sayers I. Leukotriene pathway genetics and Nat Genet. 2010;42:949–60. pharmacogenetics in allergy. Allergy. 2009;64:823–39. 64. Lu Y, Day FR, Gustafsson S, Buchkovich ML, Na J, Bataille V, et al. New loci 43. Ro M, Kim S, Pyun JA, Shin C, Cho NH, Lee JY, et al. Association between for body fat percentage reveal link between adiposity and cardiometa- arachidonate 5-lipoxygenase-activating protein (ALOX5AP) and lung bolic disease risk. Nat Commun. 2016;7:10495. function in a Korean population. Scand J Immunol. 2012;76:151–7. 65. Lam A, Rosov A, Shirak A, Seroussi E, Gootwine E. Genetic variation in 44. Goot H, Gootwine E. Wool traits of Awassi and Assaf and their respec- Afec-Assaf ewes differing in their lamb survival rate at birth. J Anim Sci. tive Booroola Merino F1 crosses. In Major genes for reproduction 2013;91(E-Suppl 2):295. in sheep. Elsen JM, Bodin L, Thimonier J, editors. Proceedings of the 66. Legaz E, Álvarez I, Royo LJ, Fernández I, Gutiérrez JP, Goyache F. Genetic International Workshop Les Colloques de l’INRA: 16–18 July 1990; Paris. relationships between Spanish Assaf (Assaf.E) and Spanish native dairy 1991. p. 341–7. sheep breeds. Small Rumin Res. 2008;80:39–44. 45. Garcia-Gamez E, Reverter A, Whan V, McWilliam SM, Arranz JJ, Kijas J. 67. Fariello MI, Boitard S, Naya H, SanCristobal M, Servin B. Detecting signa- Using regulatory and epistatic networks to extend the findings of a tures of selection through haplotype differentiation among hierarchi- genome scan: identifying the gene drivers of pigmentation in Merino cally structured populations. Genetics. 2013;193:929–41. sheep. PLoS One. 2011;6:e21158. 68. Ciani E, Crepaldi P, Nicoloso L, Lasagna E, Sarti FM, Moioli B, et al. 46. Goot H, Bor A, Hasdai A, Zenou A, Gootwine E. Body and carcass Genome-wide analysis of Italian sheep diversity reveals a strong geo- composition of Awassi, Assaf, Booroola X Awassi and Booroola X Assaf graphic pattern and cryptic relationships between breeds. Anim Genet. ram lambs. In Major genes for reproduction in sheep. Elsen JM, Bodin 2014;45:256–66. L, Thimonier J, editors. Proceedings of the International Workshop Les 69. Zhu C, Fan H, Yuan Z, Hu S, Zhang L, Wei C, et al. Detection of selection Colloques de l’INRA: 16–18 July 1990; Paris. 1991. p. 335–40. signatures on the X chromosome in three sheep breeds. Int J Mol Sci. 47. Moradi MH, Nejati-Javaremi A, Moradi-Shahrbabak M, Dodds KG, 2015;16:20360–74. McEwan JC. Genomic scan of selective sweeps in thin and fat tail sheep 70. Moioli B, Scata MC, Steri R, Napolitano F, Catillo G. Signatures of breeds for identifying of candidate regions associated with fat deposi- selection identify loci associated with milk yield in sheep. BMC Genet. tion. BMC Genet. 2012;13:10. 2013;14:76. 48. Wang H, Zhang L, Cao J, Wu M, Ma X, Liu Z, et al. Genome-wide specific 71. Garcia-Gámez E, Gutiérrez-Gill B, Sahana G, Sánchez JP, Bayón Y, Arranz selection in three domestic sheep breeds. PLoS One. 2015;10:e0128688. JJ. GWA Analysis for milk production traits in dairy sheep and genetic 49. Wang X, Zhou G, Xu X, Geng R, Zhou J, Yang Y, et al. Transcriptome support for a QTN influencing milk protein percentage in the LALBA profile analysis of adipose tissues from fat and short-tailed sheep. Gene. gene. PLoS One. 2012;7:e47782. 2014;549:252–7. 72. Rupp R, Senin P, Sarry J, Allain C, Tasca C, Ligat L, et al. A point mutation 50. Aali M, Moradi-Shahrbabak M, Moradi-Shahrbabak H, Sadeghi M. in suppressor of Cytokine Signalling 2 (Socs2) increases the suscepti- Detecting novel SNPs and breed-specific haplotypes at calpastatin bility to inflammation of the mammary gland while associated with gene in Iranian fat- and thin-tailed sheep breeds and their effects on higher body weight and size and higher milk production in a sheep protein structure. Gene. 2014;537:132–9. model. PLoS Genet. 2015;11:e1005629. 51. Bakhtiarizadeh MR, Moradi-Shahrbabak M, Ebrahimie E. Underly- 73. Rowe S, McEwan JC, Hickey SM, Anderson RA, Hyndman D, Young E, ing functional genomics of fat deposition in adipose tissue. Gene. et al. Genomic selection as a tool to decrease greenhouse gas emission 2013;521:122–8. from dual purpose New Zealand sheep. In Proceedings of the 10th 52. Raadsma HW, Jonas E, Fleet MR, Fullard K, Gongora J, Cavanagh CR, World Congress of Genetics Applied to Livestock Production: 17–22 et al. QTL and association analysis for skin and fibre pigmentation in August 2014; Vancouver. 2014. sheep provides evidence of a major causative mutation and epistatic 74. Zhang L, Liu J, Zhao F, Ren H, Xu L, Lu J, et al. Genome-wide associa- effects. Anim Genet. 2013;44:547–59. tion studies for growth and meat production traits in sheep. PLoS One. 53. Gratten J, Beraldi D, Lowder BV, McRae AF, Visscher PM, Pemberton 2013;8:e66569. JM, et al. Compelling evidence that a single nucleotide substitution 75. McRae KM, McEwan JC, Dodds KG, Gemmell NJ. Signatures of selection in TYRP1 is responsible for coat-colour polymorphism in a free-living in sheep bred for resistance or susceptibility to gastrointestinal nema- population of Soay sheep. Proc Biol Sci. 2007;274:619–26. todes. BMC Genomics. 2014;15:637. 54. Li MH, Tiirikka T, Kantanen J. A genome-wide scan study identifies a 76. Riggio V, Pong-Wong R, Sallé G, Usai MG, Casu S, Moreno CR, et al. single nucleotide substitution in ASIP associated with white versus A joint analysis to identify loci underlying variation in nematode non-white coat-colour variation in sheep (Ovis aries). Heredity (Edinb). resistance in three European sheep populations. J Anim Breed Genet. 2014;112:122–31. 2014;131:426–36. Seroussi et al. Genet Sel Evol (2017) 49:19 Page 10 of 10

77. Riggio V, Matika O, Pong-Wong R, Stear MJ, Bishop SC. Genome-wide 88. White SN, Mousel MR, Herrmann-Hoesing LM, Reynolds JO, Leymaster association and regional heritability mapping to identify loci underlying KA, Neibergs HL, et al. Genome-wide association identifies multiple variation in nematode resistance and body weight in Scottish Blackface genomic regions associated with susceptibility to and control of ovine lambs. Hered (Edinb). 2013;110:420–9. lentivirus. PLoS One. 2012;7:e47829. 78. Al-Mamun HA, Kwan P, Clark SA, Ferdosi MH, Tellam R, Gondro C. 89. Mömke S, Kerkmann A, Wöhlke A, Ostmeier M, Hewicker-Trautwein M, Genome-wide association study of body weight in Australian Merino Ganter M, et al. A frameshift mutation within LAMC2 is responsible for sheep reveals an orthologous region on OAR6 to human and bovine Herlitz type junctional epidermolysis bullosa (HJEB) in black headed genomic regions affecting height and weight. Genet Sel Evol. mutton sheep. PLoS One. 2011;6:e18943. 2015;47:66. 90. Pickering NK, Auvray B, Dodds KG, McEwan JC. Genomic prediction 79. Kemper KE, Daetwyler HD, Visscher PM, Goddard ME. Comparing link- and genome-wide association study for dagginess and host internal age and association analyses in sheep points to a better way of doing parasite resistance in New Zealand sheep. BMC Genomics. 2015;16:958. GWAS. Genet Res (Camb). 2012;94:191–203. 91. Mucha S, Bunger L, Conington J. Genome-wide association study of 80. Elbeltagy AR, Kim ES, Aboul-Naga AM, Rischkowsky B, Rothschild MF. footrot in Texel sheep. Genet Sel Evol. 2015;47:35. Heat stress in desert sheep and goats: signatures of natural selection 92. Moioli B, D’ Andrea S, De Grossi L, Sezzi E, De Sanctis B, Catillo G, et al. and initiation of GWAS. In Proceedings of the International Plant and Genomic scan for identifying candidate genes for paratuberculosis Animal Genome XXII Conference: 10–15 January 2014; San Diego. 2014. resistance in sheep. Anim Prod Sci. 2016;56:1046–55. 81. Gholizadeh M, Rahimi-Mianji G, Nejati-Javaremi A, Jan de Koning D, 93. Suarez-Vega A, Gutierrez-Gil B, Benavides J, Perez V, Tosser-Klopp G, Jonas E. Genomewide association study to detect QTL for twinning rate Klopp C, et al. Combining GWAS and RNA-Seq approaches for detec- in Baluchi sheep. J Genet. 2012;93:489–93. tion of the causal mutation for hereditary junctional epidermolysis 82. Gholizadeh M, Rahimi-Mianji G, Nejati-Javaremi A. Genomewide bullosa in sheep. PLoS One. 2015;10:e0126416. association study of body weight traits in Baluchi sheep. J Genet. 94. Zhao X, Onteru SK, Piripi S, Thompson KG, Blair HT, Garrick DJ, et al. In a 2015;94:143–6. shake of a lamb’s tail: using genomics to unravel a cause of chondro- 83. Ren X, Yang GL, Peng WF, Zhao YX, Zhang M, Chen ZH, et al. A genome- dysplasia in Texel sheep. Anim Genet. 2012;43(Suppl 1):9–18. wide association study identifies a genomic region for the polycerate 95. Atlija M, Arranz JJ, Martinez-Valladares M, Gutierrez-Gil B. Detection and phenotype in sheep (Ovis aries). Sci Rep. 2016;6:21111. replication of QTL underlying resistance to gastrointestinal nematodes 84. Kijas JW, Ortiz JS, McCulloch R, James A, Brice B, Swain B, et al. Genetic in adult sheep using the ovine 50 K SNP array. Genet Sel Evol. 2016;48:4. diversity and investigation of polledness in divergent goat populations 96. Norris BJ, Whan VA. A gene duplication affecting expression of the using 52 088 SNPs. Anim Genet. 2013;44:325–35. ovine ASIP gene is responsible for white and black sheep. Genome Res. 85. Garcia-Gámez E, Reverter A, Whan V, McWilliam SM, Arranz JJ, Inter- 2008;18:1282–93. national Sheep Genomics Consortium, et al. Using regulatory and 97. Yang GL, Fu DL, Lang X, Wang YT, Cheng SR, Fang SL, et al. Mutations in epistatic networks to extend the findings of a genome scan: identify- MC1R gene determine black coat color phenotype in Chinese sheep. ing the gene drivers of pigmentation in Merino sheep. PLoS One. Sci World J. 2013;2013:675382. 2011;6:e21158. 98. Shungin D, Winkler TW, Croteau-Chonka DC, Ferreira T, Locke AE, Magi 86. Demars J, Fabre S, Sarry J, Rossetti R, Gilbert H, Persani L, et al. Genome- R, et al. New genetic loci link adipose and insulin biology to body fat wide association studies identify two novel BMP15 mutations respon- distribution. Nature. 2015;518:187–96. sible for an atypical hyperprolificacy phenotype in sheep. PLoS Genet. 99. Schleinitz D, Bottcher Y, Bluher M, Kovacs P. The genetics of fat distribu- 2013;9:e1003482. tion. Diabetologia. 2014;57:1276–86. 87. Hazard D, Moreno C, Foulquié D, Delval E, Francois D, Bouix J, et al. 100. Cristancho AG, Lazar MA. Forming functional fat: a growing Identification of QTLs for behavioral reactivity to social separation and understanding of adipocyte differentiation. Nat Rev Mol Cell Biol. humans in sheep using the OvineSNP50 BeadChip. BMC Genomics. 2011;12:722–34. 2014;15:778.

Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research

Submit your manuscript at www.biomedcentral.com/submit